Effects of Glucagon and Insulin on Plasma Glucose, Triglyceride, and ...

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and TG-rich lipoprotein concentrations were determined. (Key words: glucagon, insulin, polyunsaturated fatty acids, n-3 and n-6 fatty acids, laying hens).

Effects of Glucagon and Insulin on Plasma Glucose, Triglyceride, and Triglyceride-Rich Lipoprotein Concentrations in Laying Hens Fed Diets Containing Different Types of Fats ´ . Bartos,† and G. Kova´cs† L. Pa´l,*,†,1 R. Grossmann,* K. Dublecz,† F. Husve´th,† L. Wa´gner,† A *Department of Functional Genomics and Bioregulation, Federal Agricultural Research Center (FAL), Institute for Animal Science and Animal Behaviour, D-29223 Celle, Germany; and †Department of Animal Physiology and Animal Nutrition, Georgikon Faculty, University of Veszprem, Deak F. Str.16., H-8360 Keszthely, Hungary ABSTRACT The influence of dietary fat supplementations differing in the ratio of n-6 to n-3 polyunsaturated fatty acids (PUFA) on the effects of glucagon and insulin on plasma glucose, triglyceride (TG), and TG-rich lipoprotein concentrations was investigated in laying hens. Birds were fed either a low-fat control diet (LF) or diets supplemented with 4% pumpkin seed oil (PO; rich in n6 PUFA) or 4% cod liver oil (CO; rich in n-3 PUFA). After 4 wk feeding of the experimental diets, hens were implanted with wing vein catheters and injected with porcine glucagon (20 µg/kg BW) and porcine insulin (0.5 IU/kg BW), 2 to 5 h after oviposition. Plasma glucose, TG, and TG-rich lipoprotein concentrations were determined

from 10 min pre-injection to 60 min post-injection. PO diet resulted in a prolonged plasma glucose response to glucagon administration and altered hypoglycemic response to insulin. However, CO diet did not influence plasma glucose response to either glucagon or insulin administration compared to LF diet. The effects of glucagon and insulin on plasma TG and TG-rich lipoproteins were similar for all diets regardless of the amount or type of fat. The results suggest that feeding dietary fats with high n-6 to n-3 PUFA ratio alters the glucagon and insulin sensitivity of plasma glucose in laying hens. Fats rich in n3 PUFA seem to have no influence on the plasma glucose response to glucagon and insulin.

(Key words: glucagon, insulin, polyunsaturated fatty acids, n-3 and n-6 fatty acids, laying hens) 2002 Poultry Science 81:1694–1702

Carbohydrate and lipid metabolism of laying birds are fundamentally different from those in mammals. They maintain a “chronic hyperglycemic” blood glucose concentration and have to meet the high demands of lipid synthesis and transport due to egg formation. It is well established that insulin and glucagon play a key role in the control of carbohydrate and lipid metabolism in avian species as well (Simon, 1989; Cogburn, 1991). There are, however, considerable differences in the regulatory role of these pancreatic hormones in birds. In contrast to mammals, glucagon is the dominant pancreatic hormone in order to meet the requirements of the constant high-carbohydrate supply for metabolic processes (Hazelwood, 2000). Several studies showed that a single administration of glucagon increased plasma glucose concentration either in laying hens (Mitchell and Raza, 1986) and cocks (Morita et al., 1987) or in turkey hens (McMurtry et al., 1996). Glucagon inhibits hepatic lipogenesis (Collado and Tasaki, 1983; Leclercq et

al., 1988; Rosebrough and McMurtry, 1992) and stimulates lipolysis in adipose tissue, thereby elevating plasma nonesterified fatty acid concentration (Morita et al., 1987; McMurtry et al., 1996). Glucagon treatment of isolated chick liver cells inhibited fatty acid synthesis (Allred and Roehrig, 1972). Plasma triglyceride (TG) was decreased by glucagon infusion in female turkeys fed ad libitum, but remained unchanged in animals deprived of feed (Kurima et al., 1994). Insulin was shown to play a secondary role in the regulation of metabolic pathways mentioned above (Hazelwood, 2000). Hypoglycemic effect of insulin was demonstrated by many studies using intravenous or intramuscular hormone injections to several species of birds (Nir and Levy, 1973; Morita et al., 1987; Leclercq et al., 1988; Neubert and Gu¨rtler, 1996). In contrast to the fact that birds are relatively resistant to high doses of insulin (Hazelwood and Lorenz, 1959; Simon, 1989), a persistent hypoglycemic state can be achieved by continuous insulin infusion (Akiba et al., 1999; Chida et al., 2000). In addition to the regulation of carbohydrate metabolism, insulin was shown to stimu-

2002 Poultry Science Association, Inc. Received for publication February 6, 2002. Accepted for publication June 7, 2002. 1 To whom correspondence should be addressed: [email protected]

Abbreviation Key: CO = cod liver oil diet; LDL = low density lipoproteins; LF = low-fat diet; PO = pumpkin seed oil diet; PUFA = polyunsaturated fatty acids; TG = triglycerides; VLDL = very low density lipoproteins.

INTRODUCTION

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DIETARY FATS AND EFFECTS OF GLUCAGON AND INSULIN IN LAYING HENS

late hepatic fatty acid production both in vivo (GomezCapilla et al., 1980) and in vitro experiments (BurghelleMayeur et al., 1989). On the other hand, very low density lipoprotein-triglyceride (VLDL-TG) secretion in the liver was suppressed by insulin administration (Mooney and Lane, 1981). Whereas insulin inhibits lipid mobilization from mammalian adipose tissues, it is not antilipolytic in birds (Hazelwood, 2000). In mammals, polyunsaturated fatty acids (PUFA) are potent regulators of carbohydrate and lipid metabolism (Field et al., 1990; Power and Newsholme, 1997). They can influence the actions of pancreatic hormones (Storlien et al., 1991), and their beneficial effects have been shown in the prevention and management of diseases typical of Western societies such as hypertension, diabetes, cardiovascular diseases etc. (Pschierer et al., 1995; Harris, 1996; Knapp, 1996). The two classes of PUFA (n-6 and n-3 fatty acids) are metabolically distinct, and in this respect n-3 PUFA appear to be more effective than fatty acids of the n-6 family (Clarke, 2000). A lower dietary ratio for n-6 to n-3 fatty acids is associated with improved glucose uptake and insulin sensitivity of skeletal muscle tissue (Storlien et al., 1997). These health-promoting effects appear to be due to the enhancement of membrane fluidity of skeletal muscle cells by highly unsaturated n-3 fatty acids (C20:5n-3, C22:6n-3), more favorable translocation, and increased intrinsic-activity of the glucose transporter GLUT-4 (Clandinin et al., 1993; Hansen et al., 1998). Dietary fish oil supplementation decreased the incorporation of fatty acids into TG and secretion of hepatic VLDL-TG compared with those observed in the low-fat (LF) diet group, but did not influence the sensitivity of hepatic VLDL-TG secretion to inhibition by insulin (Baker and Gibbons, 2000). Despite the consumption of n-3 PUFA-rich diets, increasing proportion of n-6 PUFA can lead to profound insulin resistance (Storlien et al., 1991). In addition, dietary n-6 PUFA may affect the glucagon- and β-adrenergic adenylate cyclase activity in different tissues (Dax et al., 1990; Nicolas et al., 1991). The manipulated diets of laying hens may contain sources of n-3 PUFA that can be deposited into egg yolk, providing a n-3 PUFA-rich functional food for humans (Gao and Charter, 2000; Lewis et al., 2000). Little is known, however, about the regulatory role of dietary PUFA on the insulin and glucagon actions in laying birds. Therefore, the aim of the present study was to investigate the effects of exogenous insulin and glucagon in vivo on plasma glucose, TG and TG-rich lipoprotein concentrations in laying hens fed diets containing different amounts and types (n-6 vs. n-3 PUFA) of fat.

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Lohmann Co., Cuxhaven, Germany. AMT Aromando Medizintechnik GmbH, Du¨sseldorf, Germany. 4 WDT Co., Garbsen, Germany. 5 I-5523, Sigma Chemical Co., St. Louis, MO. 6 G-3157, Sigma Chemical Co., St. Louis, MO. 7 Sigma procedure no. 315; Sigma Chemical Co., St. Louis, MO. 8 Reanal procedure no. 31951-2-99-80; Reanal Finechemical Co., Budapest, Hungary. 9 Kontron, Zurich, Switzerland. 3

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MATERIALS AND METHODS Animals and Diets Laying hens of the Lohmann Brown strain2 at 37 wk of age were housed in individual laying cages. The room temperature was maintained at 20 C, and a lighting schedule of 14 h light to 10 h darkness was provided. Diets were formulated according to the requirements recommended by the breeder company. Birds were fed either a LF control diet containing 0.5% soybean oil or diets supplemented with 4% pumpkin seed oil (PO) or 4% cod liver oil (CO). In order to make diets isocaloric, PO and CO diets contained sand as a diluting component. Hens were randomly assigned to each dietary group (n = 15) and given free access to water and feed. Composition and nutrient content of the experimental diets are presented in Table 1. The fatty acid profile of the diets is shown in Table 2. Egg weight and feed consumption were measured daily, whereas BW was recorded weekly before starting hormone treatments.

Experimental Design and Treatments After 4 wk feeding of the experimental diets, five hens from each experimental group were randomly selected and implanted with wing vein catheters3 (inside diameter, 0.762 mm; outside diameter, 1.651 mm). During the insertion of catheters into the vena cutanea ulnaris, hens were anesthetized by Narcoren4 (0.4 mL/kg BW). Porcine insulin5 at a dose of 0.5 IU/kg BW and porcine glucagon6 at a dose of 20 µg/kg BW diluted in 1 mL sterile isotonic saline were administered through the catheter. Sterile isotonic saline (1 mL/animal) was used as sham treatment. The same hens were used for sham and both hormone treatments within a dietary group.Two to three days elapsed between sham, insulin, and glucagon injections for the same hen. Blood samples (0.5 mL) of birds fed ad libitum were taken using 50 µL 0.2 M EDTA/mL of blood as anticoagulant, 2 to 5 h after the oviposition with the following intervals: 10 min before injection and at 0, 10, 20, 30, and 60 min post-injection. Blood samples were immediately chilled on ice and centrifuged at 4,000 × g for 20 min, and plasma samples were stored at 2 to 4 C before determination of plasma glucose, TG, and TG-rich lipoproteins (VLDL and LDL). All biochemical analyses were performed within 6 h after blood collection.

Biochemical Assays Plasma glucose concentration was determined by the glucose oxidase method (Trinder, 1969). Total plasma TG concentration was measured by the glycerol-3-phosphate oxydase-peroxydase method. Analyses were conducted with plasma glucose7 and total plasma TG8 reagents and an Uvikon 725 spectrophotometer.9 The absorbance of the VLDL and LDL lipoproteins were measured by the method of Griffin and Whitehead (1982). This method is based on the fact that heparin and Mg2+ selectively precipitate plasma VLDL and LDL because of their apolipoprotein B content.

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TABLE 1. Composition and calculated analysis of experimental diets Diet1 Ingredients and analysis

LF

PO

CO

Maize Wheat Maize gluten meal 60 Soybean meal 44 Starch (Maize) Added fat2 Dicalcium phosphate Limestone Salt L-Lysine DL-Methionine Antioxidants3 Vitamin premix4 Mineral premix5 Choline chloride Sand Calculated analysis ME (kcal/kg) CP Crude fiber Crude fat Lysine Methionine Methionine + cystine Calcium Total phosphorus Available phosphorus Sodium

44.40 12.00 10.00 13.60 8.43 0.50 1.50 8.60 0.50 0.10 0.10 0.02 0.05 0.10 0.10 ...

(%) 44.40 12.00 10.00 13.60 ... 4.00 1.50 8.60 0.50 0.10 0.10 0.04 0.05 0.10 0.10 4.91

44.40 12.00 10.00 13.60 ... 4.00 1.50 8.60 0.50 0.10 0.10 0.04 0.05 0.10 0.10 4.91

2,775 17.00 2.35 3.17 0.73 0.44 0.76 3.53 0.57 0.37 0.21

2,787 17.00 2.35 6.67 0.73 0.44 0.76 3.53 0.57 0.37 0.21

2,787 17.00 2.35 6.67 0.73 0.44 0.76 3.53 0.57 0.37 0.21

1,2 LF = low-fat diet supplemented with 0.5% soybean oil; PO = diet supplemented with 4% pumpkin seed oil; CO = diet supplemented with 4% cod liver oil. 3 Loxidan TD 100: LF = 0.15 g/kg; PO, CO = 0.40 g/kg. Supplied by Lohmann Animal Health, Cuxhaven, Germany. Cuxavit E50 (dl-α-tocopheryl acetate, 500 IU/g): LF = 0.01 g/kg; PO, CO = 0.04 g/kg. Supplied by Lohmann Animal Health. 4 Vitamin premix (Rovimix 428) provided the following per kilogram of feed: vitamin A, 11,000 IU (retinyl acetate); vitamin D3, 3,000 IU; vitamin E, 40 IU (dl-α-tocopheryl acetate); vitamin K3, 3 mg; riboflavin, 6 mg; dcalcium-panthotenate, 10 mg; thiamin, 2.5 mg; niacin, 30 mg; pyridoxine, 4 mg; folic acid, 1 mg; vitamin B12, 0.02 mg. Supplied by Hoffmann La Roche, Inc., Basel, Switzerland. 5 Mineral premix (Cimbria) provided the following per kilogram of feed: zinc, 60 mg; iron, 45 mg; manganese, 90 mg; copper, 6 mg; cobalt, 0.15 mg; iodine, 1 mg; selenium, 0.2 mg. Supplied by Deutsche Vilomix, Neuenkirchen bei Osnabru¨ck, Germany.

It provides the relative comparison of TG-rich lipoprotein (VLDL and LDL) contents in plasma samples. Total fat content of diets was extracted, and fatty acids were methylated by Association of Official Analytical Chemists methods (Association of Official Analytical Chemists, 1990a,b). The fatty acid methyl esters were separated and analyzed by means of gas chromatography in a Carlo Erba HRGC 5300 Mega Series chromatograph10 equipped with an Omegawax 320 capillary column.11 Identification of individual fatty acids was made using a standard mixture of fatty acid methyl esters (PUFA-2).12

Statistical Analysis Statistical analysis was carried out by three-way ANOVA using feeding group, hormonal treatment, and time as main 10

Carlo Erba Strumentazione SA, Rodano (MI), Italy. Supelco catalogue 24152; Supelco, Bellefonte, PA. 12 Supelco catalogue 4-7015-U; Supelco, Bellefonte, PA. 13 StatSoft Inc., Tulsa, OK. 11

effects. Significant differences were tested by the least significant difference test (Snedecor and Cochran, 1967). All statistical analyses were conducted using the Statistica 5.0 statistical package.13 Data for glucose concentrations were transformed logarithmically before analysis to achieve homogeny of variances. Results are expressed as untransformed means ± SEM.

RESULTS Performance Parameters The values for egg weight, egg production, feed intake, and BW of laying hens are presented in Table 3. Differences in fat content and fatty acid composition of experimental diets failed to result in differences (P ≥ 0.05) in the performance parameters (egg weight, egg production, feed intake, and BW of the hens) after the 4-wk long feeding period.

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DIETARY FATS AND EFFECTS OF GLUCAGON AND INSULIN IN LAYING HENS TABLE 2. Fatty acid composition and content of experimental diets Diet1 Fatty acid type

LF

C14:0 C16:0 C16:1n-7 C18:0 C18:1n-9 C18:2n-6 C18:3n-3 C20:1n-9 C20:4n-6 C20:5n-3 C22:5n-3 C22:6n-3 SAT2 MUFA3 Total n-6 Total n-3 PUFA4 n-6 to n-3 ratio

0.12 14.57 0.21 2.72 24.64 52.40 3.15 0.23 0.54 ... ... ... 17.41 25.08 52.94 3.15 56.09 16.80

C18:2n-6 C18:3n-3 C20:5n-3 C22:6n-3 Total n-6 Total n-3

15.78 0.95 ... ... 15.94 0.95

PO

CO

(weight % of total fatty acids) 0.11 12.40 0.18 4.29 32.12 48.47 1.15 0.14 0.21 ... ... ... 16.80 32.44 48.68 1.15 49.83 42.33 (g/kg diet) 30.71 0.73 ... ... 30.85 0.73

2.97 13.88 4.11 2.65 21.86 27.38 2.05 3.58 0.54 3.71 1.10 5.18 20.96 29.55 27.92 12.04 39.96 2.31 17.35 1.30 2.35 3.28 17.69 7.63

1 LF = low-fat diet supplemented with 0.5% soybean oil; PO = diet supplemented with 4% pumpkin seed oil; CO = diet supplemented with 4% cod liver oil. 2 SAT = saturated fatty acids. 3 MUFA = monounsaturated fatty acids. 4 PUFA = polyunsaturated fatty acids.

TABLE 3. Egg weight, egg production, feed intake, and BW of laying hens during the 4-wk period prior to the hormone treatments1 Weeks of the trial Performance parameter

Dietary group

2

wk 1

Egg weight

LF PO CO

59.1 ± 0.9 59.9 ± 1.4 59.7 ± 1.0

Egg production

LF PO CO

86.7 ± 2.2 87.6 ± 1.5 91.3 ± 2.4

Feed intake

LF PO CO

118.9 ± 2.9 117.1 ± 4.6 115.1 ± 2.8

BW

LF PO CO

1,801 ± 35 1,755 ± 68 1,754 ± 46

wk 2

wk 3

(g/egg) 59.5 ± 0.9 59.7 ± 1.1 60.3 ± 1.3 60.2 ± 1.5 59.5 ± 0.8 59.9 ± 0.7 (%) 97.0 ± 1.0 93.3 87.6 ± 1.5 91.3 87.7 ± 1.1 89.4 (g/d/hen) 112.5 ± 2.3 116.7 112.5 ± 3.3 114.2 115.3 ± 2.5 116.0 (g/hen) 1,826 ± 35 1,804 ± 57 1,809 ± 42

wk 4 59.9 ± 1.1 60.4 ± 1.4 60.2 ± 0.7

± 2.0 ± 0.8 ± 1.3

95.1 ± 1.3 90.6 ± 1.9 92.3 ± 1.8

± 3.5 ± 2.7 ± 3.3

118.2 ± 4.1 117.1 ± 3.0 117.1 ± 4.2

1,836 ± 39 1,830 ± 56 1,832 ± 41

1,843 ± 43 1,837 ± 51 1,842 ± 43

1 Values are means ± SEM of daily (egg weight, egg production, and feed intake) or weekly (BW) observations for individual hens (n = 15). 2 LF = low-fat diet supplemented with 0.5% soybean oil; PO = diet supplemented with 4% pumpkin seed oil; CO = diet supplemented with 4% cod liver oil.

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FIGURE 1. Effects of glucagon (20 µg/kg BW) and insulin (0.5 IU/kg BW) administration on plasma glucose concentrations of laying hens fed a low-fat diet (LF; 䊏), or diets supplemented with 4% pumpkin seed oil (PO; high proportion of C18:2n-6; 䊊) or 4% cod liver oil (CO; high proportion of long-chain n-3 fatty acids; 䉭) for 4 wk. Values are means ± SEM, n = 5 (sham, PO: n = 4). The asterisks indicate a significant difference (P ≤ 0.05) compared to basal values (0 min). Means at the same time with different letters are significantly different (P ≤ 0.05).

Plasma Glucose Plasma glucose concentrations in the pre- and post-injection periods are illustrated in Figure 1. There were no differences (P ≥ 0.05) among dietary groups in plasma glucose levels before starting treatments with exogenous hormones or sham (−10 and 0 min). Plasma glucose concentrations did not differ after injection of isotonic saline. Glucagon administration increased plasma glucose concentration in all dietary groups. It was significant (P ≤ 0.05) the first 10 min after the injection in the LF and PO groups, and at 20 min post-injection in the CO group. The maximum levels of glucose were found in each group at 20 min posttreatment. In the LF and CO groups these peak levels returned to pre-injection level within 60 min after treatment. However, plasma glucose concentration of the PO group was still higher (P ≤ 0.05) at 60 min post-injection than those measured at the same sampling time for the two other groups or the basal value of this dietary group (0 min). Exogenous insulin resulted in a decrease of plasma glucose. The glucose concentrations measured at 10, 20, and 30 min after the insulin administration in the LF, and at 20 or 30 min post-injection in the CO group were lower (P ≤ 0.05) compared with glucose concentrations measured at the time of injection (0 min). The minimum values in these two groups were obtained at 20 min after the insulin administration, and plasma glucose reached the pretreatment (0 min) levels by the time 60 min post-injection. In the PO group, however, plasma glucose concentration showed a different response curve from those of the other two dietary groups. It decreased (P ≤ 0.05) during the first 20 min after the insulin treatment (0 min) and then increased during the next 10 min and showed a significant (P ≤ 0.05) decline at 60 min in comparison to pretreatment value at 0 min.

After insulin administration, plasma glucose level of hens fed different diets did not show any significant difference when comparing the values at the same sampling times.

Plasma Triglycerides The plasma TG response of laying hens to glucagon and insulin administration is shown in Figure 2. Pretreatment TG concentrations (−10 and 0 min) were not different (P ≥ 0.05) among dietary groups. The dietary regimen did not influence the responses of plasma TG to hormone and saline injections. Although glucose concentrations were significantly altered, plasma TG levels were not affected (P ≥ 0.05) by glucagon or insulin treatment until the end of the sampling period.

Plasma TG-Rich Lipoproteins Similarly to plasma TG, plasma TG-rich lipoproteins (VLDL and LDL) also failed to show any significant changes (P ≥ 0.05) before and after insulin, glucagon, or saline administrations in all dietary groups (Figure 3). The plasma VLDL and LDL absorbance values estimated turbidimetrically correlated positively with TG concentrations measured by enzymatic kits (r = 0.95, P ≤ 0.05; 0.85, P ≤ 0.05; and 0.89, P ≤ 0.05 for LF, PO, and CO diets, respectively).

DISCUSSION The performance of the hens was not significantly affected by the type of lipid supplements to the diet, showing that the three groups were in a similar physiological state. The supplementation of diet with 4% maize oil, providing 22.4 g linoleic acid/kg diet increased the egg weight in the study of Whitehead et al. (1993). The authors suggested the

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FIGURE 2. Effects of glucagon (20 µg/kg BW) and insulin (0.5 IU/kg BW) administration on plasma triglyceride concentrations of laying hens fed a low-fat diet (LF; 䊏), or diets supplemented with 4% pumpkin seed oil (PO; high proportion of C18:2n-6; 䊊) or 4% cod liver oil (CO; high proportion of long-chain n-3 fatty acids; 䉭) for 4 wk. Values are means ± SEM, n = 5 (sham, PO: n = 4).

positive effect of dietary linoleic acid on plasma estradiol metabolism, which may enhance the lipid and protein synthesis for egg formation. However, in our experiment no differences were observed in the egg weight using LF, PO, and CO diets containing 15.8, 30.8, and 17.3 g/kg linoleic acid, respectively. Other authors also failed to show any effect of PUFA-rich diets on egg weight using soybean oil (Shafey et al., 1992) or fish oil (Meluzzi et al., 2000). These conflicting results could be due to the use of different strains, age, dietary fat type, inclusion rates, and duration of the experiment. The inclusion of fish oil (Huang et al., 1990) or corn oil (Edwards and May, 1965) had no effect

on feed consumption or live weight as compared to LF control diets. These observations are consistent with our results. Before hormone treatments and in sham-treated hens, plasma glucose concentrations were in the normal physiological range and were not affected by the dietary regimen. Porcine glucagon and insulin were chosen to test the effects of these pancreatic hormones. Chicken and porcine glucagon were found to have similar metabolic clearance rates and elicited similar biological responses in the female turkey hen (McMurtry et al., 1996). Furthermore, no speciesspecific insulin and glucagon receptors were shown in birds

FIGURE 3. Effects of glucagon (20 µg/kg BW) and insulin (0.5 IU/kg BW) administration on plasma triglyceride-rich lipoprotein absorbance of laying hens fed a low-fat diet (LF; 䊏), or diets supplemented with 4% pumpkin seed oil (PO; high proportion of C18:2n-6; 䊊) or 4% cod liver oil (CO; high proportion of long-chain n-3 fatty acids; 䉭) for 4 wk. Values are means ± SEM, n = 5 (sham, PO: n = 4).

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(Kemmler et al., 1978; Hazelwood, 2000). The homologous insulin is, however, a more potent regulator of plasma glucose than the heterologous bovine or porcine one (Hazelwood, 2000). A well-known finding is that glucagon injection induces plasma glucose peaks (Mitchell and Raza, 1986; Morita et al., 1987; Brenes et al., 1989). Decreased plasma glucose levels after insulin administration in the domestic fowl have also been reported by many previous studies (Morita et al., 1987; Saadoun et al., 1988; Neubert and Gu¨rtler, 1996). Our study is a pioneer work in studying the influences of different dietary PUFA supplementations on the metabolic effects of insulin and glucagon in vivo in the domestic fowl. These data show that the type of the additional dietary fat (n-6 to n-3 PUFA ratio) influenced the response of plasma glucose to exogenous glucagon and insulin. Hens fed diets supplemented with 4% pumpkin seed oil, containing a high level of linoleic acid (C18:2n-6) showed prolonged plasma glucose response to glucagon administration compared with hens fed LF or CO diets. However, the magnitudes of the hyperglycemic effect after glucagon treatment were similar for all dietary groups. Few studies in mammals have focused on the investigations of the relationship between metabolic actions of glucagon and dietary PUFA. Diets deficient in essential fatty acids led to decreased glucagonstimulated adenylate cyclase activity in rats (Brivio-Haugland et al., 1976). Nicolas et al. (1991) found a higher βadrenergic receptor affinity in adipocytes of pigs fed a diet containing sunflower oil (high in C18:2n-6) than in the control animals fed diets without fat supplementation. Furthermore, in rat liver membranes, increased glucagon- and isoproterenol- (β-agonist) stimulated adenylate cyclase activities were demonstrated if the unsaturation level of fatty acids was increased by corn oil supplementation (Dax et al., 1990). The authors suggested that the diet-induced alterations may act at the receptor level (Dax et al., 1990). These studies are in agreement with our results showing that an increase in the proportion of dietary n-6 to n-3 PUFA may improve responsiveness of plasma glucose to glucagon. Similarly, the PO-feeding also altered the plasma glucose response to insulin in our experiment. Although the maximum rate of insulin-induced decrease in plasma glucose was not different among dietary groups, insulin administration led to different time-course changes of plasma glucose in the PO group as compared to those observed in the hens fed either the LF or CO diets containing lower n6 to n-3 PUFA ratios. A possible explanation for this finding may be the higher dietary intake of n-6 PUFA, thus, the increase in the n-6 to n-3 PUFA ratio, which can alter the insulin sensitivity. In addition, exogenous insulin administration can provoke glucagon release from the pancreas to normalize the hypoglycemia caused by insulin (Hazelwood and Langslow, 1978; Sitbon and Mialhe, 1978). This glucagon response, which can modify insulin action diversely, could also have played a role in our study. In contrast, the CO, high in long-chain n-3 PUFA, failed to affect insulin sensitivity in terms of plasma glucose levels in comparison to LF diet. The results of several studies conducted by mammals using the hyperinsulinemic, euglycemic clamp

technique support our observations. These findings clearly demonstrate the importance of the fatty acid composition of dietary fats on insulin action. In rats, diets high in saturated, monounsaturated fatty acids or n-6 PUFA caused a profound insulin resistance while rats fed diets high in longchain n-3 PUFA maintained normal insulin action (Storlien et al., 1991). According to the data of Storlien et al. (1996), a high n-6 to n-3 PUFA ratio related directly to insulin resistance in humans, too. Our data support the idea that dietary n-6 to n-3 PUFA ratio can alter plasma glucose response to both glucagon and insulin in laying hens. No effects of the dietary fats on the plasma TG and TG-rich lipoprotein concentrations were observed. Dietary PUFA, particularly those derived from fish oils, inhibited lipid synthesis and enhanced hepatic fatty acid oxidation, thus leading to a decrease in circulating blood TG in mammals (Clarke, 2000). The study of Castillo et al. (1999) showed that supplementation of 10% menhaden oil to the diet can produce a significant hypotriglyceridemia in chicks. According to our results, hepatic TG synthesis of laying hens seems to be less sensitive to the fatty acid composition of the diet compared to that of chicks. In the present study, plasma TG and TG-rich lipoprotein levels remained unchanged after glucagon and insulin injections over the 60-min sampling period, when significant changes of plasma glucose concentration occurred. Applying a ten times higher dose of insulin (5 IU/kg BW) than in our experiment, Nir and Levy (1973) also showed that insulin did not influence plasma TG concentration in cockerels and geese 1 h after the injection, although changes of plasma glucose were detected. The same treatment, however, provoked a substantial rise in plasma TG 3 h posttreatment in geese. Numerous reports showed both in vitro and in vivo in rats, that insulin inhibits the secretion of hepatic VLDL and apolipoprotein B. Similar effects of the hormone were also reported in vitro in birds. Very high concentrations of insulin can stimulate lipoprotein lipase activity in chicken adipose tissue (Borron et al., 1979). Chirieac et al. (2000) suggested that the inhibition of the hepatic VLDL release allows intestinal TG to clear from the circulation during the postprandial period. Thus, insulin may prevent the competition of lipoproteins of hepatic and intestinal origin for common clearance pathways after feeding. Interestingly, insulin is not able to regulate the secretion of VLDL-TG in hepatocytes derived from insulin-resistant rats (Bourgeois et al., 1995), in obese women (Lewis et al., 1993) and in patients with type 2 diabetes (Malmstrom et al., 1997). Birds are known as insulin-resistant animals compared to mammals (Hazelwood, 2000). In addition, in laying hens insulin probably fails to supress hepatic VLDL and apolipoprotein B secretion because of the requirement of the growing oocyte for TG. In contrast to our findings with ad libitum fed laying hens, glucagon inhibited apolipoprotein B secretion in hepatocytes derived from fed- and fasted rats (Bjornsson et al., 1992; Chirieac et al., 2000). In our study the method of Griffin and Whitehead (1982) was used for lipoprotein analysis. This method measures the relative changes of apolipoprotein B-containing lipoproteins in the plasma. Li-

DIETARY FATS AND EFFECTS OF GLUCAGON AND INSULIN IN LAYING HENS

poproteins of intestinal origin, termed portomicrons in birds, also contain apolipoprotein B (Hermier, 1997). The portomicrons, however, reach the liver before the peripheral tissues because of the poorly developed lymphatic system in avian species. It is likely that portomicrons are partly metabolized by the avian liver (Surai, 1999). Furthermore, extrahepatic tissues have a very rapid catabolism of portomicrons released from the liver (Hermier, 1997). Hence, they are usually in very low amounts in the circulation (Hermier, 1997), probably with low contribution to TG-rich lipoprotein levels measured in the present study. We observed very high individual variations in plasma TG and TG-rich lipoprotein levels in the sampling period. This may be explained by differences in plasma estrogen concentration which, according to Walzem et al. (1999), fluctuates during the oviposition cycle and acts as a main regulator of hepatic VLDL secretion in laying hens. The plasma TG content originates mainly from TG-rich lipoproteins explaining the positive correlations for all dietary groups observed between the results of the two different methods for TG and TG-rich lipoprotein determination. In conclusion, the dietary n-6 to n-3 PUFA ratio modulates the plasma glucose response to glucagon and insulin in laying hens. Fat-supplemented diets rich in n-6 PUFA may lead to higher glucagon sensitivity and can alter responsiveness to insulin. Dietary oils high in long-chain n3 PUFA do not modify the effect of glucagon and insulin on plasma glucose compared to a LF diet. Our data suggest that dietary PUFA and glucagon or insulin administration do not affect the plasma TG and TG-rich lipoprotein levels in the same period as it could be detected in the case of plasma glucose.

ACKNOWLEDGMENTS This research was supported by L. Pal’s scholarship from the German Academic Exchange Service (DAAD) and OTKA T-37963. The authors express appreciation to Dr. I. Halle and Dr. A. Berk (Federal Agricultural Research Center, Braunschweig, Germany) for their help and support. Furthermore, the authors wish to thank the technical staff of both departments for excellent assistance during this study.

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